Each trajectory was subjected to a set of at least 32 analyses. Broadly the 32 analyses can be summarized as the analysis of gross structural changes (RMSD), assessment of secondary structural changes and calculation of number of contacts between residues, solvent accessible surface area (SASA) and Ramachandran distributions of residues. The full set includes, but is not limited to, the calculation of the following properties as a function of simulation time: radius of gyration, end-to-end distance, CONGENEAL [1] dissimilarity score to starting structure, Cα-RMSD to starting structure, Cα-RMSD of residues involved in secondary structure, main-chain (MC) SASA, side-chain (SC) SASA, polar SASA, non-polar SASA, MC polar SASA, SC polar SASA, MC non-polar SASA, SC non-polar SASA, total SASA, total native contacts, native MC-to-MC contacts, native MC-to-SC contacts, native SC-to-SC contacts, non-native MC-to-MC contacts, non-native MC-to-SC contacts, non-native SC-to-SC contacts, total non-native contacts, intra-molecular polar contacts, intra-molecular hydrophobic contacts, intra-molecular polar-to-non-polar atom contacts, by residue contacts, by residue type contacts, helical content (by ψ/φ), beta content (by ψ/φ), extended content (by ψ/φ), other content (i.e. not in standard ψ/φ definitions), secondary structure assignment by dictionary of secondary structures of proteins (DSSP) [2], and Pardi et al helical content [3].  SASA was calculated according to the algorithm of [4] with a probe radius of 1.4 Å. Contacts are considered only between heavy atoms. Native contacts are those present in the minimized starting structure. Unless polar or non-polar is specified, a contact is defined as two carbon atoms whose separation distance is less than 5.4 Å or two non-carbon or one carbon and one non-carbon atoms whose separation distance is less than 4.6 Å. A polar contact is between two atoms whose absolute charges are greater than 0.3 charge units (q) and whose separation distance is less than 4.6 Å. A non-polar contact is between two atoms whose absolute charges are less than 0.3 q and separation distance is less than 5.4 Å. A polar to non-polar contact is one between two atoms, one with an absolute charge greater than 0.3 q and one whose absolute charge is less than 0.3 q and a separation distance less than 5.4 Å. All analyses were performed in ilmm.

Where NMR data are available from the PDB or the BioMagResBank (BMRB) [5] in formats that could be directly employed for analyses or readily converted by the DOCR tools [6], trajectories were analyzed in the context of those data. For the purposes of comparison to nuclear Overhauser effects (NOE), an experimental NOE was considered satisfied if the r^-6 weighted distance of the closest pairs of protons specified by the constraint was less than 5.0 Å or the experimental upper-bound. Order parameters (S²), primarily in the form of main-chain amide, were calculated from MD trajectories as described in [7] and an R to the experimental values was used as a measure of agreement. Similarly, chemical shifts predicted from MD trajectories were compared to the experimental values by the measure of R. Residual dipolar couplings  (RDC) from MD trajectories were calculated by the algorithm presented in [8] and compared with experimental data. All analyses were performed in ilmm with the exception of chemical shifts, which were calculated with SHIFTS [9].

References

1. Yee, D.P. and K.A. Dill, Families and the structural relatedness among globular proteins. Protein Sci, 1993. 2(6): p. 884-99.
2. Kabsch, W. and C. Sander, Dictionary of Protein Secondary Structure - Pattern-Recognition of Hydrogen-Bonded and Geometrical Features. Biopolymers, 1983. 22(12): p. 2577-2637.
3. Pardi, A., M. Billeter, and K. Wuthrich, Calibration of the Angular-Dependence of the Amide Proton-C-Alpha Proton Coupling-Constants, 3jhn-Alpha, in a Globular Protein - Use of 3jhn-Alpha for Identification of Helical Secondary Structure. Journal of Molecular Biology, 1984. 180(3): p. 741-751.
4. Lee, B. and F.M. Richards, Interpretation of Protein Structures - Estimation of Static Accessibility. Journal of Molecular Biology, 1971. 55(3): p. 379-&.
5. Doreleijers, J.F., S. Mading, D. Maziuk, K. Sojourner, L. Yin, J. Zhu, J.L. Markley, and E.L. Ulrich, BioMagResBank database with sets of experimental NMR constraints corresponding to the structures of over 1400 biomolecules deposited in the Protein Data Bank. Journal of Biomolecular Nmr, 2003. 26(2): p. 139-146.
6. Doreleijers, J.F., A.J. Nederveen, W. Vranken, J.D. Lin, A.M.J.J. Bonvin, R. Kaptein, J.L. Markley, and E.L. Ulrich, BioMagResBank databases DOCR and FRED containing converted and filtered sets of experimental NMR restraints and coordinates from over 500 protein PDB structures. Journal of Biomolecular Nmr, 2005. 32(1): p. 1-12.
7. Wong, K.B. and V. Daggett, Barstar has a highly dynamic hydrophobic core: Evidence from molecular dynamics simulations and nuclear magnetic resonance relaxation data. Biochemistry, 1998. 37(32): p. 11182-11192.
8. Losonczi, J.A., M. Andrec, M.W.F. Fischer, and J.H. Prestegard, Order matrix analysis of residual dipolar couplings using singular value decomposition. Journal of Magnetic Resonance, 1999. 138(2): p. 334-342.
9. Osapay, K. and D.A. Case, A New Analysis of Proton Chemical-Shifts in Proteins. Journal of the American Chemical Society, 1991. 113(25): p. 9436-9444.